PsbD | D2 protein of PSII

Changes of PSII and PSI antenna and core protein levels. Proteins from control and Tl-treated white mustard leaves were separated by SDS-PAGE followed by immunodetection with antibodies against Lhcb1, Lhcb2, Lhca1 (antenna proteins) and D1, D2, CP43, PsbO, PsaA (core proteins). Samples were loaded on the equal amount of chlorophyll (0.25 ?g). Description of samples abbreviation as given in the legend to Fig. 3

Accumulation of photosynthetic complexes in the mutant strains.Cells were grown in TAP medium at 25?°C under LED white light (8??mol photons m?2?s?1) and collected at the mid-log phase. Two independent lines are shown for each construct. Protein samples were loaded on an equal chlorophyll basis (0.5??g per lane), and a dilution series of WT samples is shown for semi-quantitative comparison. Antibodies against essential subunits of PSII (D2), PSI (PsaA), cytochrome b6f (PetA) and ATP synthase (AtpB) probed the accumulation of the respective photosynthetic complexes. Numbers on the left side of the blots are molecular weights in kD. See Supplementary Fig. 9 for the uncropped blot images.

Thylakoid protein phosphorylation and state transitions are disturbed after high light treatment in pgr6 background. a Total protein extracts of 4-week-old wild type (WT), pgr6-1, pgr6?2, sps2 and stn7/stn8 analysed by immunoblotting with anti-phosphothreonine antibody; the principal thylakoid phospho proteins are indicated on the right according to their size. Core photosystem II proteins D1 (PsbA) and D2 (PsbD) are indicated as a single band due to their poor resolution. Actin was used as a loading control. b Lhcb1 and Lhcb2 phosphorylation levels were visualised after separation on Phostag™-pendant acrylamide gels. The upper band corresponds to the phosphorylated form (p-), stn7/stn8 double mutant is a non-phosphorylated control. c Average transient of the variable room temperature chlorophyll fluorescence measured during the transition from red (660nm) supplemented with far-red light (720nm) state 1 to pure red light state 2 (n?=?4 independent pots containing 2–3 plants). The fluorescence curves from pgr6 and sps2 are shifted on the x-axis to allow visualising the FMST1 and FMST2 values. The x-axis time scale refers to the wild-type curve. d Calculated quenching related to state transition (qT?=?(FMST1?–?FMST2)/FM), expressed as the percentage of FM that is dissipated by the state 1 to state 2 transition, of wild type (WT), pgr6?1 and sps2 under moderate light (120?mol?m?2?s?1) (ML) and after 3?h of high light (500?mol?m?2?s?1) (HL). Whiskers and box plot shows the minimum, first quartile, median, average, third quartile and maximum of each dataset (n?=?4 biologically independent samples); p-values are calculated via a two-tailed Student’s t test. e STN7 phosphorylation level visualised after separation on Phostag™-pendant acrylamide gels. The upper band corresponds to the phosphorylated form (p-), a protein sample from stn7/stn8 double mutant was loaded as a control for the antibody specificity. Uncropped images of the membranes displayed in a, b and e are available as Supplementary Fig. 11. Data points for items c, d are available as Supplementary data 2

Photomorphogenesis is not significantly affected in ros1, nrpd1, nrpe1, rdr2 or ago4 mutant seedlings. (a) Phenotypes (upper panel) and the corresponding maximum quantum yields of PSII (Fv/Fm) (lower panel) of 4-day-old etiolated seedlings. Fv/Fm was measured with an imaging Chl fluorometer (Imaging PAM). Scale bar?=?1 cm. (b) Phenotypes (upper panel) and corresponding Fv/Fm values (lower panel) of 3-day-old etiolated seedlings which had been exposed to continuous light for 24 h. (c) Immunoblot analysis of the PSII core proteins (D1 and D2), Lhcb1 and FLU during greening of etiolated seedlings. WT seedlings were grown for 3 days in the dark and exposed to light for between 0 and 48 h, as indicated. Extracted total proteins were normalized with respect to fresh weight and fractionated by SDS-PAGE. Blots were then probed with antibodies raised against the individual proteins. Total proteins from 5-day-old WT seedlings grown under continuous light (LL) and LD conditions (LD) were used as positive controls. The total protein accumulation of each sample was visualized by staining the gel with Coomassie Blue R250 (C.B.). The figure was assembled from different blots (delineated by a black rectangle) and full-length blots are presented in Supplementary Fig. S7. (d) Immunoblot analysis of representative photosynthesis proteins of 3-day-old etiolated mutant seedlings which had been exposed to continuous light for 24 h. Immunoblot analysis was performed as in (c). The figure was assembled from different blots (delineated by a black rectangle) and full-length blots are presented in Supplementary Fig. S8. (e) Real-time PCR analyses of 3-day-old etiolated WT (Col-0) and mutant seedlings that had been exposed to continuous light for 24 h. Real-time PCR was performed with primers specific for the nuclear genes LHCB1.2, LHCB2.1, LHCB6, LHCA5, PSBP-1 and PSBTn, and the plastid genes psaA and atpB. Note that the primers for LHCB2.1 also amplify LHCB2.2 mRNA. Expression values are reported relative to the corresponding transcript levels in the WT and were normalized with respect to the expression level of ACTIN2. Data are shown as mean values?±?SD from three different plant pools.

Immunoblot analysis of the photosynthetic proteins on the basis of equal total Ponceau S dye stained blot with proteins from leaves of wild type plants and pap1 mutant grown on MS medium in Petri dishes for four weeks under a 16 h light/8 h dark photoperiod at 23 °C with 100 ?E m?2 s?1. Proteins were visualized by immunoblotting using antibodies specific for RbcL, PsaB, PsbD, AtpB, RpoB, AccD and Lhcb2.4 proteins.

Accumulation dynamics of photosynthesis-related proteins during de-etiolation.Three-day-old etiolated seedlings of Arabidopsis thaliana were illuminated for 0 hr (T0), 4 hr (T4), 8 hr (T8), 12 hr (T12), 24 hr (T24), 48 hr (T48), 72 hr (T72), and 96 hr (T96) under white light (40 µmol/m2/s). (A) Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane and immunodetected with antibodies against PsbA, PsbD, PsbO, PetC, PsaD, PsaC, Lhcb2, AtpC, ELIP, POR, phyA, HY5, and ACTIN proteins. (B–C) Quantification of PsbA, PetC, and PsaC during de-etiolation. Heatmap (B) was generated after normalization of the amount of each protein relative to the last time point (T96). Graph (C) corresponds to the absolute quantification of proteins at T96. Error bars indicate ± SD (n = 3). Quantification of photosystem-related proteins during de-etiolation is detailed in Figure 6—figure supplement 1.Figure 6—source data 1.Quantitative data for immunoblot analysis.Quantitative data for immunoblot analysis.Quantification of photosynthesis-related proteins.(A) Immunodetection of PsbA, PetC, and PsaC during de-etiolation. Dilutions were used for the later time points to avoid saturation of the signal. (B) Different bands were detected by Amersham Imager program and quantified by Image QuantTL (Amersham). (C) Calibration curves were created using recombinant proteins (Agrisera). Calibration curve composition: PsbA 10 ng (A; lane a), 5 ng (b), 2.5 ng (c), and 1.25 ng (d); PetC 10 ng (e), 5 ng (f), 2.5 ng (g), and 1.25 ng (h); PsaC 3 ng (i), 1.5 ng (l), 0.75 ng (m), and 0.325 ng (n). Data indicate mean ± SD (n = 3–4). Raw data and calculations are shown in Figure 6—source data 1.